The genetic variation at 12 isozyme loci was investigated in the three species (Xanthium italicum, X. strumarium, and X. orientale) forming a X. strumarium complex in Italy. Very little variation was found within species at the loci studied in contrast to the considerable interspecies genetic differentiation at several loci. The gene differentiation between species was ranged from 61 to 91%. The observed genetic structure of the X. strumarium complex was consistent with that found for predominantly autogamous species. The values of maximum outcrossing rates estimated in original sampling sites and in a field test ranged from 8 to 17%, confirming previous observations that Xanthium species are predominantly self-pollinated. Gene duplications were evident in the three Xanthium species because of their likely polyploid origin. The percentage of duplicate loci exhibiting “fixed heterozygosity” was 25, 25, and 16% in X. italicum, X. strumarium, and X. orientale, respectively. Data presented supported that both mating system and ploidy level were fundamental features in adaptation process of investigated Xanthium species. Some evidence suggested that polyploidization occurred before speciation of X. italicum, X. strumarium, and X. orientale. As a consequence, a common ancestral progenitor could be postulated for the three species. During geographical adaptation, the three species fixed alternative alleles in some loci, and the process was favored by the predominantly autogamous mating system. On the contrary, fixed heterozygosity in duplicated loci allowed maintenance of a sufficient level of gene diversity in the three Xanthium species to ensure wide adaptability in different microhabitats (i.e., abandoned land, roadsides, and field crops) and to avoid the negative effect of inbreeding depression.